Legionella pneumophila, the etiologic agent of Legionnaires' disease, is a significant cause of both community-acquired and nosocomial pneumonia. This gram-negative bacterium is an intracellular parasite of human alveolar macrophages and aquatic protozoa. Although critical for intracellular survival, the mechanisms used by L. pneumophila for iron acquisition have remained largely unknown. However, recent biochemical and genetic studies have uncovered four Legionella strategies for iron uptake. First, a potentially novel siderophore was discovered in the supernatants of virulent strains. The detection of this iron chelator required the universal CAS assay and was strain and culture dependent, explaining the previous negative results. Second, they identified an iron-regulated gene that encodes an analog of aerobactin synthetases and promotes intracellular replication, suggesting the existence of a second L. pneumophila siderophore. Third, the analysis of a mutant that was defective for intracellular infection and virulence revealed a gene (ira216) encoding a di/tripeptide transporter. The reduced ability of this mutant to acquire intracellular iron suggests that L. pneumophila imports iron-loaded peptides during macrophage infection. Finally, another iron acquisition and infectivity mutant appears to lack an ABC- transporter (ira225), which is similar, but not identical, to other bacterial systems that assimilate unchelated iron. This grant renewal will define more precisely the nature of these pathways. Upon demonstration that the siderophores are expressed within infected host cells, a set of mutants will be used to ascertain the role of the iron chelators in intraphagosomal growth. Mass spec and NMR will define the structure of the purified siderophores. CAS and Csaky assays as well as Southern hybridizations will determine whether these iron scavengers are peculiar to the pathogenic Legionella species, as preliminary data suggest. Next ira216 and ira225 mutants will be assessed for reduced abilities to grow in defined media where iron is provided as either iron-loaded peptides or ferric citrate. Conversely, the cloned genes will be examined for the abilities to enhance the iron assimilation of E. coli uptake mutants. To gauge the total number of iron uptake systems that operate intracellularly, double mutants, e.g. Sid-, ira216 negative strain, will be analyzed for iron and infectivity defects. Taken together, the proposed experiments will offer greater insight into Legionella pathogenesis as well as intracellular parasitism and microbial iron acquisition in general.